CN108753790B - BAVM-associated gene markers and mutations thereof - Google Patents
BAVM-associated gene markers and mutations thereof Download PDFInfo
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- CN108753790B CN108753790B CN201810598796.3A CN201810598796A CN108753790B CN 108753790 B CN108753790 B CN 108753790B CN 201810598796 A CN201810598796 A CN 201810598796A CN 108753790 B CN108753790 B CN 108753790B
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Abstract
The invention discloses a gene marker related to BAVM (basic immunoglobulin of human immunodeficiency virus) and a mutation thereof, wherein the gene marker is PITPNM 3. The invention discovers a new mutation c.274C > T on PITPNM3 for the first time, further proves that PITPNM3 is related to the occurrence and development of BAVM through a zebra fish experiment, and prompts that PITPNM3 can be used for clinical diagnosis of BAVM.
Description
Technical Field
The invention belongs to the field of biological medicine, and relates to a gene marker related to BAVM and mutation thereof, wherein the gene is PITPNM 3.
Background
Arteriovenous malformations (BAVM) are the major cause of juvenile idiopathic cerebral hemorrhage, accounting for 2% of total hemorrhagic stroke (Horton JC. Arteriovenous malformations of the brain. N Engl J Med, 2007; 357(17): 1774-1775.). The structure of the blood vessel comprises a malformed blood vessel mass formed by winding abnormal blood vessels, a terminal blood supply artery and a drainage vein. The main clinical manifestations of BAVM are also epilepsy and focal neurological dysfunction, with mortality associated with cerebral hemorrhage. BAVM spontaneous cerebral hemorrhage often recurs, and as the number of bleeding increases, symptoms and signs can escalate, or even become life-threatening. Statistically, the mortality rate caused by BAVM is about 10% -15%, and the permanent re-residual rate is about 20% -30%.
With the continuous improvement of the overall level of the medical career in China, advanced imaging diagnosis technologies such as DSA and MRI and the like are gradually popularized, and the effective detection of clinical BAVM cases is objectively and greatly promoted, especially for some asymptomatic patients.
In terms of treatment, although most BAVMs can be cured by surgical resection, interventional embolization or stereotactic radiotherapy, high-grade BAVMs with malformed vascular mass diameter >3cm, focal sites in functional areas, or deep drainage veins, still have significant treatment risks according to the Spetzler and smith spring grading methods, have a high incidence of post-operative neurological dysfunction, and are not ideal for embolization and radiotherapy. It has now been found that BAVM lesions can exhibit a dynamically changing trend: lesion volumes may increase in steps, morphological and quantitative changes in the supplying and draining arteries, concomitant aneurysms, etc. (advanced tumors of the graft in adults. NJ Med [ J ],1999,340(23): 1812-1818.). If the BAVM can be found early, the pathological grade of the BAVM is possibly low, and the BAVM is easy to treat; meanwhile, most BAVM patients are asymptomatic at ordinary times, and can be discovered only after bleeding or accidentally discovered in the process of searching for the causes of epilepsy or long-term intractable headache, and the hiding property constitutes a potential threat to life and health. Both of the above mentioned two points put real requirements on early discovery and early treatment of BAVM. Because the cost of the existing clinical cerebral angiography or nuclear magnetic resonance for diagnosing BAVM is high, the clinical cerebral angiography or nuclear magnetic resonance is difficult to be used for large-scale screening of BAVM cases, a simple, convenient and cheap BAVM early diagnosis tool is found, meanwhile, the risk assessment can be provided for the probability of BAVM suffering from a crowd, and the method is the direction of the efforts of vast medical researchers. Therefore, the research on the pathophysiology changes caused by serious complications such as bleeding by exploring the etiology and mechanism of the BAVM becomes a hot spot in BAVM research.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a gene related to BAVM and a mutant thereof, thereby providing a new means for diagnosing BAVM.
The invention also provides a BAVM model and a construction method thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the invention, there is provided a nucleotide sequence of PIPPNM 3, wherein the nucleotide has a mutation at position 274 compared with SEQ ID No.1, and the mutation is c.274C > T.
Furthermore, the nucleotide sequence is shown as SEQ ID NO. 2.
In a second aspect, the present invention provides a mutant PITPNM3 protein encoded by the nucleotide sequence of the first aspect of the invention, the amino acid sequence of which has been changed from Arg to Ter at position 92, i.e. a stop codon.
A third aspect of the invention provides a kit for detecting BAVM, the kit comprising the detection reagents of PITPNM 3.
Further, the agent is selected from:
an agent that detects the level of expression of PITPNM 3; or
A reagent for detecting the 274 th nucleotide site of the PITPNM3 gene; or
And (3) a reagent for detecting the 92 nd amino acid position of the PITPNM3 protein.
Further, the reagent comprises a probe which specifically recognizes the PITPNM3 gene, or a primer which specifically amplifies the PITPNM3 gene, or an antibody or ligand which specifically binds to the PITPNM3 protein.
The fourth aspect of the invention provides a kit for detecting a PITPNM3 gene mutant, which comprises a nucleic acid probe complementary to the nucleotide sequence of the first aspect of the invention or a primer for amplifying the nucleotide sequence of the first aspect of the invention.
A fifth aspect of the invention provides a method of constructing a BAVM model by administering an inhibitor of PITPNM3 to a culture system, wherein the culture system includes, but is not limited to, a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.
Further, the culture system is an animal system, and preferably, the animal is zebra fish.
Further, the PITPNM3 inhibitor is an antisense oligonucleotide.
Preferably, the antisense oligonucleotide is a morpholine antisense oligonucleotide.
More preferably, the sequence of the morpholine antisense oligonucleotide is shown as SEQ ID NO. 3.
Further, the zebrafish embryo is a single-cell stage to two-cell stage embryo.
A sixth aspect of the invention provides the use of any one of:
a. use of a nucleotide according to the first aspect of the invention in the manufacture of a product for diagnosing BAVM;
b. use of a protein according to the second aspect of the invention in the manufacture of a product for diagnosing BAVM;
the use of PITPNM3 for the preparation of a product for diagnosing BAVM;
d. use of a kit according to a fourth aspect of the invention in the manufacture of a product for diagnosing BAVM;
e. use of a BAVM model constructed using the method of the fifth aspect of the invention for screening a candidate for treatment of BAVM;
use of PITPNM3 for the manufacture of a medicament for the treatment of BAVM.
Further, the product described in c comprises an agent for detecting the expression level of PITPNM3, wherein the agent is an agent for detecting the expression level of PITPNM 3; or a reagent for detecting mutation sites and mutants thereof;
e, if the symptoms of BAVM in the culture system are relieved after the administration of the substance to be screened, the substance to be screened is a candidate drug for treating BAVM.
The medicine in f comprises an accelerant of PITPNM3, and preferably, the accelerant is an expression vector containing PITPNM3 mRNA.
The invention has the advantages that:
the invention firstly discovers that the mutation of PITPNM3 is related to BAVM, and whether a patient has BAVM can be judged by detecting whether the mutation occurs at a specific site of PITPNM 3.
The invention discovers that the expression level of PITPNM3 is related to BAVM for the first time, and whether a patient has BAVM can be judged by detecting the expression level of PITPNM 3.
The invention provides a method for constructing a BAVM model, and provides a new means for clinical BAVM research and drug screening.
Drawings
FIG. 1 is a Sanger sequencing validation and clinical imaging diagnosis of patients carrying the PITPNM3 mutation; wherein, the A sanger sequencing map is shown in the figure, and the B clinical imaging diagnosis map is shown in the figure.
FIG. 2 is a confocal microscope fluorescence image of zebra fish cerebral vessels.
FIG. 3 is a diagram of the BAVM-like phenotype pattern of zebrafish mutants.
FIG. 4 is a graph showing the growth and RT-PCR validation of zebrafish mutants; wherein Panel A is a growth diagram and Panel B is a binding site diagram of the action site and the upstream and downstream primers of the morpholine antisense oligonucleotide; FIG. C shows RT-PCR validation of the mutants.
Figure 5 is a statistical plot of the number of zebrafish BAVM phenotypes.
Detailed Description
According to the invention, through extensive and deep research, the bleeding risk and development outcome of BAVM are predicted from the aspects of etiology and genetics by utilizing whole exome sequencing and through the comparative research of core families, sporadic cases and healthy people, and early accurate and individualized treatment is facilitated. Meanwhile, a new method and experience are provided for the etiological study of the complex polygenic cerebrovascular congenital disease.
In the present invention, the term "exon" refers to the portion of mature mRNA that is retained, i.e., the portion of mature mRNA corresponding to the gene. Introns are the parts that are spliced out during mRNA processing and are not present in mature mRNA. Both exons and introns are for genes, the encoded part is an exon, the non-encoded part is an intron, and the intron has no genetic effect.
The present invention may utilize any method known in the art for detecting genes and their encoded proteins. It will be appreciated by those skilled in the art that the means by which the gene is detected is not an important aspect of the present invention. The genes of the present invention are detected using a variety of detection techniques known to those of ordinary skill in the art, including, but not limited to: nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification technology and immunodetection technology.
Methods for detecting the level of expression of PITPNM3 include (but are not limited to): polymerase Chain Reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), Transcription Mediated Amplification (TMA), Ligase Chain Reaction (LCR), Strand Displacement Amplification (SDA) and Nucleic Acid Sequence Based Amplification (NASBA), In Situ Hybridization (ISH), microarrays, Southern or Northern blots, sandwich ELISA, Radioimmunoassays (RIA), direct, indirect or contrast enzyme-linked immunosorbent assays (ELISA), Enzyme Immunoassays (EIA), Fluorescence Immunoassays (FIA), western blots, immunoprecipitations and immunoassays based on any particle (e.g. using gold, silver or latex particles, magnetic particles or quantum dots). Among them, PCR requires reverse transcription of RNA into DNA before amplification (RT-PCR), TMA and NASBA to directly amplify RNA. As used herein, "detecting the expression level of a gene" refers to determining the presence of mRNA of a marker gene and its expression level in a biological sample in order to predict the course of BAVM and can be accomplished by measuring the amount of mRNA. Analytical methods for this purpose are, but are not limited to, RT-PCR, competitive RT-PCR, Real-time RT-PCR, RNase Protection Assay (RPA), northern blotting, DNA microarray chips, etc.
Methods for detecting gene mutations or protein mutations include (but are not limited to): PCR-SSCP method, heteroduplex analysis method, denaturation gradient gel electrophoresis method, chemical cutting mismatch method, allele specific oligonucleotide analysis method, DNA chip technology, ligase chain reaction, allele specific amplification method, direct sequencing method.
In the present invention, any suitable assay platform can be used to determine the presence of mRNA in a sample. For example, the assay may be in the form of a dipstick (dipstick), membrane (membrane), chip (chip), disk (disk), test strip (test strip), filter (filter), microsphere (microspherole), slide (slide), multiwell plate (multiwell plate) or optical fiber (optical fiber). The assay system can have a solid support to which a nucleic acid corresponding to mRNA is attached. The solid support may comprise, for example, a plastic, a silicon wafer, a metal, a resin, a glass, a membrane, a particle, a precipitate (precipitate), a gel, a polymer, a sheet (sheet), a sphere, a polysaccharide, a capillary, a film (film), a plate, or a slide. The assay components can be packaged together after manufacture as a kit for detecting mRNA.
Generally, PCR uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase the copy number of a target nucleic acid sequence; RT-PCR Reverse Transcriptase (RT) is used to prepare complementary DNA (cDNA) from mRNA, and the cDNA is then amplified by PCR to produce multiple copies of the DNA; TMA autocatalytically synthesizes multiple copies of a target nucleic acid sequence under substantially constant conditions of temperature, ionic strength and pH, wherein multiple RNA copies of the target sequence autocatalytically generate additional copies, TMA optionally including the use of blocking, partial, terminating and other modifying moieties to improve the sensitivity and accuracy of the TMA process; LCR with target nucleic acid adjacent region hybridization of two sets of complementary DNA oligonucleotides. The DNA oligonucleotides are covalently linked by DNA ligase in repeated cycles of heat denaturation, hybridization, and ligation to produce a detectable double-stranded ligated oligonucleotide product; the SDA uses multiple cycles of the following steps: primer sequence pairs anneal to opposite strands of the target sequence, primer extension in the presence of dNTP α S to produce double-stranded hemiphosphorothioated (phosphorothioated) primer extension products, endonuclease-mediated nicking of the hemimodified restriction enzyme recognition site, and polymerase-mediated extension from the 3' end of the nick to displace the existing strand and produce a strand for the next round of primer annealing, nicking and strand displacement, thereby causing geometric amplification of the products.
Nucleic acid hybridization techniques of the invention include, but are not limited to, In Situ Hybridization (ISH), microarrays, and Southern or Northern blots. In Situ Hybridization (ISH) is a hybridization of specific DNA or RNA sequences in a tissue section or section using a labeled complementary DNA or RNA strand as a probe (in situ) or in the entire tissue if the tissue is small enough (whole tissue embedded ISH). DNAISH can be used to determine the structure of chromosomes. Rnash is used to measure and locate mRNA and other transcripts (e.g., ncRNA) within tissue sections or whole tissue embedding. Sample cells and tissues are typically treated to fix the target transcript in situ and to increase probe access. The probe is hybridized to the target sequence at high temperature, and then excess probe is washed away. The localization and quantification of base-labeled probes in tissues labeled with radiation, fluorescence or antigens is performed using autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can also use two or more probes labeled with radioactive or other non-radioactive labels to detect two or more transcripts simultaneously.
As an alternative embodiment, the kit of the invention comprises a specific primer pair for amplification of PITPNM 3; a standard DNA template; and (3) PCR reaction liquid.
As a more preferable embodiment, the kit is a fluorescent quantitative PCR detection kit, and the primer is suitable for detection of SYBR Green, TaqMan probes, molecular beacons, double-hybrid probes and composite probes.
In a more preferred embodiment, the PCR reaction solution in the kit is a fluorescent quantitative PCR reaction solution, and further comprises a fluorescent dye.
In a more preferred embodiment, the fluorescent quantitative PCR reaction solution comprises dNTP and Mg2+The fluorescent dye is SYBR Green II, and the Taq enzyme is hot start enzyme.
The immunization method according to the present invention may be based on, for example, any of the following methods.
Immunoprecipitation is the simplest immunoassay method; this method measures the amount of precipitate that is formed after the reagent antibody has been incubated with the sample and reacted with the target antigen present therein to form insoluble aggregates. The immunoprecipitation can be either qualitative or quantitative.
In a particle immunoassay, multiple antibodies are attached to the particle and the particle is capable of binding many antigenic molecules simultaneously. This greatly accelerates the speed of the visible reaction. This allows for a fast and sensitive detection of the biomarker.
In immunoturbidimetry (immunonephelometry), the interaction of an antibody and a target antigen on a biomarker causes the formation of an immune complex that is too small to precipitate. However, these complexes will scatter incident light, which can be measured using a turbidimeter. The concentration of the antigen (i.e. biomarker) can be determined within a few minutes of the reaction.
Radioimmunoassay (RIA) methods use radioisotopes such as I125 to label antigens or antibodies. The isotope used emits gamma rays, which are usually measured after removal of unbound (free) radiolabel. The main advantages of RIA compared to other immunoassays are higher sensitivity, easy signal detection and confirmation, fast assay. The main disadvantages are the health and safety risks posed by the use of radiation and the time and expense associated with maintaining the licensed radiation safety and disposal procedures. For this reason, RIA has been largely replaced by enzyme immunoassays in routine clinical laboratory practice.
Enzyme Immunoassays (EIAs) have evolved as alternatives to Radioimmunoassays (RIA). These methods use enzymes to label the antibody or target antigen. The sensitivity of EIA is close to that of RIA and there is no risk caused by radioisotopes. One of the most widely used EIA methods for detection is enzyme-linked immunosorbent assay (ELISA). The ELISA method may use two antibodies, one specific for the target antigen and the other coupled to an enzyme, the addition of an enzyme substrate causing the generation of a chemiluminescent or fluorescent signal.
Fluorescence Immunoassay (FIA) refers to an immunoassay that uses a fluorescent label or an enzyme label that acts on a substrate to form a fluorescent product. Fluorescence measurements are inherently more sensitive than chromatic (spectrophotometric) measurements. Thus, the FIA method has higher analytical sensitivity than the EIA method using absorption (optical density) measurement.
Chemiluminescent immunoassays use a chemiluminescent label that produces light when excited by chemical energy; the emission is measured using a photodetector.
Thus, the immunization method according to the present invention can be carried out using well-known methods. Any direct (e.g., using a sensor chip) or indirect method may be used in the detection of the biomarkers of the invention.
The term "DNA library" refers to a mixture of DNA fragments of a certain size obtained by disrupting a desired fragment of a genome.
Methods for preparing DNA libraries are well known to those skilled in the art and include, but are not limited to, the steps of:
providing a sample to be tested, said sample containing broken double-stranded nucleic acid fragments derived from genomic DNA and said nucleic acid fragments having blunt ends;
adding an adaptor connecting sequence at the end of the double-stranded nucleic acid fragment; adding adaptors to both ends of the double-stranded nucleic acid fragment through the adaptor-joining sequence, wherein the adaptors have a primer binding region and a junction-complementary region, the junction-complementary region being complementary to the adaptor-joining sequence; the sequence of the primer binding region of the linker flanking the 3 'and 5' ends is different.
Amplifying the DNA double-stranded nucleic acid fragment with the adaptor obtained in the previous step with a first primer and a second primer, thereby obtaining a mixture of PCR amplification products, wherein the primers have an adaptor binding region corresponding to the primer binding region of the adaptor and a sequencing probe binding region located outside the adaptor binding region.
In a preferred embodiment, the cleavage product, the end-repair product, the linker product and the enrichment product may also be purified. Purification conditions and parameters are well known to those skilled in the art, and it is within the ability of those skilled in the art to make certain changes or optimizations to the reaction conditions.
The terms "exon capture" and "chip hybridization" are used interchangeably and refer to the process by which a probe specifically selects and binds to DNA fragments in the exon regions of a library.
DNA molecules are normally double stranded and therefore, prior to capture, the DNA molecule must become single stranded, typically by denaturing it by heating for melting purposes, and the melted DNA molecule is rapidly cooled, i.e., remains single stranded. The library is denatured and then subjected to capture hybridization with the chip on the hybridization platform. Molecular hybridization is carried out under stringent conditions between the DNA fragments containing the exon regions and the probes immobilized on the chip. Preferably, the concentration of probe molecules on the chip is much higher than the concentration of target molecules. After hybridization, the captured sequences are collected by methods such as denaturation and purified to obtain a mixture of sequences from the captured sequences.
In the present invention, the inhibitor of PITPNM3 refers to any substance that can decrease the activity of PITPNM3 protein, decrease the stability of PITPNM3 gene or protein, down-regulate the expression of PITPNM3 protein, decrease the effective acting time of PITPNM3 protein, or inhibit the transcription and translation of PITPNM3 gene, and these substances can be used in the present invention, for example, the inhibitor is: nucleic acid inhibitors, protein inhibitors, antibodies, ligands, proteolytic enzymes, protein binding molecules, as long as they are capable of down-regulating the expression of PITPNM3 protein or its encoding gene at the protein or gene level.
Wherein the nucleic acid inhibitor is selected from: an interfering molecule that targets PITPNM3 or its transcript and is capable of inhibiting PITPNM3 gene expression or gene transcription, comprising: shRNA (small hairpin RNA), small interfering RNA (sirna), dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming said shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid. The protein binding molecule is selected from: a substance that specifically binds to the PITPNM3 protein, such as an antibody or ligand capable of inhibiting the activity of the PITPNM3 protein.
As an alternative of the invention, the inhibitor of PITPNM3 is an antibody that specifically binds PITPNM 3. The specific antibody comprises a monoclonal antibody and a polyclonal antibody; the invention encompasses not only intact antibody molecules, but also any fragment or modification of an antibody, e.g., chimeric antibodies, scFv, Fab, F (ab') 2, Fv, etc. As long as the fragment retains the ability to bind to the PITPNM3 protein. The preparation of antibodies for use at the protein level is well known to those skilled in the art and any method may be used in the present invention to prepare such antibodies.
As an alternative embodiment of the invention, the inhibitor of PITPNM3 is a PITPNM3 specific small interfering RNA molecule. As used herein, the term "small interfering RNA" refers to a short segment of double-stranded RNA molecule that targets mRNA of homologous complementary sequence to degrade a specific mRNA, a process known as RNA interference (RNAInterferce). Small interfering RNA can be prepared as a double-stranded nucleic acid form, which contains a sense and an antisense strand, the two strands only in hybridization conditions to form double-stranded. A double-stranded RNA complex can be prepared from the sense and antisense strands separated from each other. Thus, for example, complementary sense and antisense strands are chemically synthesized, which can then be hybridized by annealing to produce a synthetic double-stranded RNA complex.
As an alternative of the present invention, the inhibitor of PITPNM3 may also be a "Small hairpin RNA (shRNA)" which is a non-coding Small RNA molecule capable of forming a hairpin structure, the Small hairpin RNA being capable of inhibiting gene expression via an RNA interference pathway. As described above, shRNA can be expressed from a double-stranded DNA template. The double-stranded DNA template is inserted into a vector, such as a plasmid or viral vector, and then expressed in vitro or in vivo by ligation to a promoter. The shRNA can be cut into small interfering RNA molecules under the action of DICER enzyme in eukaryotic cells, so that the shRNA enters an RNAi pathway. "shRNA expression vector" refers to some plasmids which are conventionally used for constructing shRNA structure in the field, usually, a "spacer sequence" and multiple cloning sites or alternative sequences which are positioned at two sides of the "spacer sequence" are present on the plasmids, so that people can insert DNA sequences corresponding to shRNA (or analogues) into the multiple cloning sites or replace the alternative sequences on the multiple cloning sites in a forward and reverse mode, and RNA after the transcription of the DNA sequences can form shRNA (short Hairpin) structure. The "shRNA expression vector" is completely available by the commercial purchase of, for example, some viral vectors.
In a preferred embodiment of the present invention, the inhibitor of PITPNM3 is an antisense oligonucleotide, which refers to an RNA or DNA fragment having a complementary sequence to an RNA sequence in vivo and capable of hybridizing to a complementary strand by base pairing, thereby affecting the transcription or translation process. More preferably, the inhibitor is a morpholine antisense oligonucleotide, which is named after a morpholine ring on its nucleotide backbone, replacing a ribonucleotide ring in RNA or a deoxyribonucleotide ring in DNA. Morpholine antisense oligonucleotide is a novel antisense oligonucleotide capable of inhibiting the splicing process of mRNA in cells, thereby inhibiting the expression of genes; meanwhile, the product has good stability, solubility and cell permeability.
The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations.
Example 1 screening for BAVM-associated genes
1. Sample collection
123 patients diagnosed with BAVM at the beijing altar hospital during 11 to 2016 (blood was retained for patients and their biological parents, core pedigree) were collected. Diagnosis of cerebral arteriovenous malformations (BAVM) was confirmed by Digital Subtraction Angiography (DSA), magnetic resonance imaging/angiography, or computed tomography, all parents underwent physical examination to rule out serious vascular problems.
Exclusion criteria were: (1) known hemorrhagic telangiectasia (HHT), Capillary malformation-AVM (CM-AVM), churge-Weber syndrome, or other mendelian inherited vascular diseases; or (2) incomplete clinical data.
After exclusion, 100 were eligible, and both patients and their normal clinical phenotype biological parents underwent WES.
The study was approved by the ethical committee of the Beijing Tiantan hospital. The patient or his parent sign informed consent. The zebrafish experiment was approved by the university of Beijing animal protection and use Committee (IACUC).
2. Whole exon sequencing and mutation annotation
1) Extraction of DNA
The whole genome DNA of peripheral blood of patients and parents is extracted by using an Axygen blood genome DNA extraction kit, and the DNA is quantitatively detected by using 1% agarose gel electrophoresis and a Nanodrop 2000 spectrophotometer.
2) Construction of DNA libraries
The genomic DNA was randomly fragmented into fragments of 200-300bp, using an ultrasonic disrupter (Covaris 2, Massachusetts, USA), 500ng of the purified DNA fragment was subjected to end repair and ligated with a labeled linker to construct a DNA sequencing library. The 3-step enzymatic reaction was performed according to the illumina standard library construction method: the sample library was formed by end repair, addition of "a" and ligation of Illumina sequencing adaptors (by PCR reaction, an 8bp barcode was ligated to the DNA fragment).
3) Exon capture
Exon capture was accomplished using SureSelect Human All Exon V6+ UTR r2core design (91Mb, Agilent), the specific procedure is described in the instructions.
4) Sequencing and analysis
Sequencing was performed using Illumina HiSeq 4000 platform, SNP sites and indel markers were detected using Genome Analysis Toolkit 3.4.0 and haplotypecall, annotation was performed using puch, and new, complex and stealth genetic mutations were calculated using Gemini (version 0.19.1). The conservation and pathogenicity of candidate variations are predicted using bioinformatic prediction tools. All variants were screened for pathogenic variants against the public database 1000 genes Project (http:// www.internationalgenome.org /), exterior variant server, NHLBI GO Exterior Sequencing Project (ESP) (http:// EVS. gs. Washington. edu/EVS /), and exterior Aggregation Consortium (ExAC) (http:// exterior. Broadantintitute. org /).
3. Results
The results show that the patient's PITPNM3 gene shows the mutation c.274c > T (p.arg92ter) compared to the reference transcript (NM — 031220.3).
Example 2 validation of mutation sites Using sanger sequencing
1. Genomic DNA obtained from patients and parents was purified using the Axygen-AP-GX-50 kit and Sanger sequenced on an ABI3730 sequencer.
2. Results
The results are shown in FIG. 1, and the sequencing results show that nucleotide 274 of PITPNM3 in the patient has changed from C to T.
Example 3 zebra fish validation
1. Zebra fish strain
Using Tg (kdrl.4: mCherry)pku6Transgenic zebrafish experiments were conducted and the expression of mCherry red fluorescence in vivo was regulated by kdrl.4, an endothelial cell-specific gene. Zebrafish and embryos were grown according to standard. To avoid fungal infection, embryos were incubated in 0.5g/L methylene blue. 1-phenyl-2-thiourea (0.003%) was used to inhibit pigmentation.
2. In vitro transcription and embryo injection
To verify in vivo that antisense morpholine antisense oligonucleotides, MO1 (for exon 5-intron 5 splicing region), MO2 (for the AUG site), were designed against pintpnm 3 homologous to zebrafish, the specific sequences are as follows:
MO1:5'-AAAGTCGCTTACTTACTTTGACACA-3'(SEQ ID NO.3)
MO2:5'-CCCTCTGGTCTCTTTAGCCATCCTG-3'(SEQ ID NO.4)
control MO: 5'-CCTCTTACCTCAGTTACAATTTATA-3' (SEQ ID NO.5)
The gene knock-down reagent Morpholino 5ng was injected into the cells from the unicellular stage to the bicellular stage of zebrafish embryos.
3. Fluorescence observation
The 48h zebrafish were stripped of the egg membranes using forceps and anesthetized using Tricane. Embryos were fixed in low-melt glue, photographed using a confocal microscope, and zebrafish cerebrovascular phenotypes were assessed using a blinding method by two researchers.
4. RNA extraction
Total RNA from 24h embryos was extracted using Trizol method, and the tissues or cells were homogenized and added to 1.5ml centrifuge tubes. Chloroform of 0.2 times the same volume was added, and the mixture was shaken and mixed for 30 seconds. Centrifuge at 15000g for 3min at room temperature. The supernatant was transferred to another clean centrifuge tube. Equal volume of isopropanol was added to the supernatant and mixed on a shaker for 30 s. Centrifugation at 15000g for 5min at room temperature resulted in RNA pellet formation on the side of the bottom of the centrifugation and the supernatant was carefully aspirated off. Cleaning: 1ml of 75% ethanol was added to the centrifuge tube containing the RNA pellet, and the mixture was mixed on a shaker for 30 seconds. Centrifuge at 15000g for 1min at room temperature. The supernatant was carefully aspirated. The washing step is repeated. The centrifuge tube was inverted on filter paper to dry the RNA.
5. Reverse transcription
The RNA product was subjected to Reverse transcription using the OneScript Reverse Transcriptase kit, and the Reverse transcription product was subjected to PCR amplification. The primer sequences are as follows:
the primer sequences of PITPNM3 are as follows:
a forward primer: 5'-ATGGGACACATCGACCAAACA-3' (SEQ ID NO.6)
Reverse primer: 5'-TTGCGACGGCATCTTGGTAG-3' (SEQ ID NO.7)
And (3) PCR reaction conditions: 94 ℃ for 2min, (94 ℃ for 30s, 60 ℃ for 30s, 72 ℃ for 1.5min) x 25-30 cycles
6. Statistical analysis
All experiments were performed in at least three replicates and statistical analysis was performed using SPSS 15.0 software. Statistically significant is defined as P < 0.05. Zebra fish phenotypic ratio data were analyzed using the t-test.
7. Results
Immunofluorescence analysis of zebra fish cerebral vessels revealed a series of BAVM characteristics: (1) abnormal posterior circulation connecting segments (PCS) extend and fuse with the primary hindbrain channel (PHBC) near the Basal Communicating Artery (BCA), forming a semilunar malformation representation; (2) expanding the BCA and the PCS; (3) dysplasia of the anterior cerebral artery (fig. 2).
Further verifying the function of the PITPNM3 gene and its mutation, the PITPNM3 mutant (the zebrafish with the PITPNM3 knocked down) showed edema of various degrees around the head and heart (FIG. 4A), while the RT-PCR pattern showed that the PITPNM3 transcript of about 218bp was truncated (FIG. 4C).
Statistics of the number of zebrafish with BAVM phenotype showed that the number of zebrafish with BAVM was significantly increased in the PITPNM3 knockout group compared to the control group (fig. 5), indicating that PITPNM3 is associated with occurrence and development of BAVM.
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.
Sequence listing
<110> Neuko department of neurosurgery research in Beijing
PEKING UNION MEDICAL College HOSPITAL CHINESE ACADEMY OF MEDICAL SCIENCES
<120> BAVM-associated Gene marker and mutation thereof
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2925
<212> DNA
<213> Homo sapiens
<400> 1
atggccaagg cgggccgtgc aggtggtcct cccccgggcg gcggtgcccc ctggcacctt 60
cgaaatgtcc tcagtgactc tgtggagagc tcagatgatg aattctttga tgccagagag 120
gagatggctg aagggaagaa tgccatcctc attgggatga gccagtggaa ctccaatgac 180
ctcgtggagc agatcgagac catggggaaa ctggacgagc atcaaggaga agggaccgcg 240
ccgtgcacat ccagcatcct ccaggagaag cagcgagaac tgtaccgggt ttccttgaga 300
agacagaggt tcccagccca gggaagcatc gagatccacg aagacagcga ggaaggctgc 360
ccgcagcgct cctgcaagac acatgtcctc ctgctggtcc tgcatggggg aaacatcctg 420
gacacgggtg ccggggaccc gtcctgcaag gcagccgaca tccacacctt cagctccgtg 480
ctggagaagg tcacacgagc ccatttccct gctgccctgg gccacatcct catcaagttc 540
gtcccctgtc ctgccatctg ctctgaggct ttctcgcttg tctctcacct gaacccctac 600
agccacgatg agggctgcct cagcagcagc caggaccacg tccctctggc cgcccttccc 660
ctgttggcca tctcctcccc gcagtaccag gatgctgtcg ccaccgtcat cgagcgagcc 720
aaccaggtct acagagagtt cctgaagtcc tctgatggga ttggcttcag tgggcaggtg 780
tgtctcatcg gggactgtgt ggggggcctc ctggccttcg atgccatctg ctacagtgcg 840
gggccctcag gggacagccc tgccagcagc agccggaagg ggagcatcag cagcacccag 900
gacaccccag tcgcggtgga ggaagattgc agcctggcca gcagcaagcg tctcagcaaa 960
agcaacattg acatctccag tgggttggag gatgaggagc ccaagaggcc gttgccgcgg 1020
aaacagagcg actcctccac ctatgactgc gaggccatca cccagcacca tgccttcctc 1080
tcaagcatcc actccagcgt gctaaaggat gagtctgaga ccccggcggc tggggggccg 1140
cagctccctg aggtcagcct gggccgcttt gacttcgatg tgtccgactt cttcctcttc 1200
ggctcgccac tgggcctggt cctggccatg cggaggacgg tgctgcctgg gctggacggc 1260
ttccaggtgc gtcctgcctg cagccaggtc tacagcttct tccattgcgc agacccctct 1320
gcctcacggc tcgagccact gctggagccc aagttccacc tggtgccgcc tgtcagcgtg 1380
cctcgctacc agaggttccc actgggcgat gggcagtccc tcctcctcgc tgatgcccta 1440
cacacccaca gccccctctt cctggagggc agctcccggg acagcccgcc acttctggat 1500
gcccctgcct cgccccctca ggcctcgagg ttccagcgcc caggacggag gatgagcgag 1560
gggagctccc acagcgagag ctcggagtcc tcggacagca tggcacccgt gggtgcctcc 1620
cgcatcacag ccaagtggtg gggaagcaag aggatcgact atgccctgta ctgccctgat 1680
gtcctcacgg ccttccccac cgtggccctg ccccacctct tccacgccag ttactgggag 1740
tccacagacg tggtggcctt catcctgaga caggtaatgc gctatgagag cgtgaacatc 1800
aaggaaagcg cccgcctgga ccctgcagca ctgagtcctg ccaacccccg ggagaagtgg 1860
cttcgtaagc ggactcaggt caagctgagg aatgtcacgg ctaatcaccg ggccaatgat 1920
gtgattgctg ctgaagatgg cccccaggtc ctggtggggc ggttcatgta cgggcccctc 1980
gacatggtgg ctctgactgg agagaaggtg gacatcctag taatggcaga gccatcctca 2040
ggccgctggg tacacctgga cacagagatc accaacagca gtggtcgcat cacatacaat 2100
gtgccgcggc cccggcgcct gggggttggt gtctatcctg tgaagatggt cgtcaggggc 2160
gaccagacct gtgccatgag ctacctcacg gtgttgccca ggggcatgga gtgtgtagtg 2220
ttcagcattg atgggtcctt cgcggccagc gtgtctatca tgggaagcga ccccaaggtc 2280
cggccgggtg cagtggatgt tgtccggcac tggcaggact tgggctacat gatcctttac 2340
atcacgggac ggccggacat gcagaagcag cgggtggtgt cgtggctgtc ccagcacaac 2400
ttcccacagg gcatgatctt cttctccgat gggctggtgc atgacccgct gcggcagaag 2460
gccatcttcc tgcgcaacct catgcaggag tgcttcatca aaatcagtgc ggcctatggc 2520
tccacgaagg acatctctgt ctacagcgtg ctgggcctgc ctgcctccca gatcttcatt 2580
gtgggccggc ccaccaagaa gtaccaaacc cagtgccagt tcctgagcga gggctacgcc 2640
gcacacctgg ccgcgctgga ggccagccac cgctcacgcc caaagaagaa caactcgcgc 2700
atgatcctgc gcaagggcag cttcgggctg cacgcgcagc cagagttcct gcggaagcgc 2760
aaccacctgc gcagaaccat gtcagtgcag cagcccgacc cgcccgccgc caaccccaag 2820
cccgagcggg cccagagcca gcccgagtcg gacaaagacc acgagcggcc gctgccggcg 2880
ctcagctggg cgcgtgggcc ccccaagttc gagtcggtgc cctga 2925
<210> 2
<211> 2925
<212> DNA
<213> Homo sapiens
<400> 2
atggccaagg cgggccgtgc aggtggtcct cccccgggcg gcggtgcccc ctggcacctt 60
cgaaatgtcc tcagtgactc tgtggagagc tcagatgatg aattctttga tgccagagag 120
gagatggctg aagggaagaa tgccatcctc attgggatga gccagtggaa ctccaatgac 180
ctcgtggagc agatcgagac catggggaaa ctggacgagc atcaaggaga agggaccgcg 240
ccgtgcacat ccagcatcct ccaggagaag cagtgagaac tgtaccgggt ttccttgaga 300
agacagaggt tcccagccca gggaagcatc gagatccacg aagacagcga ggaaggctgc 360
ccgcagcgct cctgcaagac acatgtcctc ctgctggtcc tgcatggggg aaacatcctg 420
gacacgggtg ccggggaccc gtcctgcaag gcagccgaca tccacacctt cagctccgtg 480
ctggagaagg tcacacgagc ccatttccct gctgccctgg gccacatcct catcaagttc 540
gtcccctgtc ctgccatctg ctctgaggct ttctcgcttg tctctcacct gaacccctac 600
agccacgatg agggctgcct cagcagcagc caggaccacg tccctctggc cgcccttccc 660
ctgttggcca tctcctcccc gcagtaccag gatgctgtcg ccaccgtcat cgagcgagcc 720
aaccaggtct acagagagtt cctgaagtcc tctgatggga ttggcttcag tgggcaggtg 780
tgtctcatcg gggactgtgt ggggggcctc ctggccttcg atgccatctg ctacagtgcg 840
gggccctcag gggacagccc tgccagcagc agccggaagg ggagcatcag cagcacccag 900
gacaccccag tcgcggtgga ggaagattgc agcctggcca gcagcaagcg tctcagcaaa 960
agcaacattg acatctccag tgggttggag gatgaggagc ccaagaggcc gttgccgcgg 1020
aaacagagcg actcctccac ctatgactgc gaggccatca cccagcacca tgccttcctc 1080
tcaagcatcc actccagcgt gctaaaggat gagtctgaga ccccggcggc tggggggccg 1140
cagctccctg aggtcagcct gggccgcttt gacttcgatg tgtccgactt cttcctcttc 1200
ggctcgccac tgggcctggt cctggccatg cggaggacgg tgctgcctgg gctggacggc 1260
ttccaggtgc gtcctgcctg cagccaggtc tacagcttct tccattgcgc agacccctct 1320
gcctcacggc tcgagccact gctggagccc aagttccacc tggtgccgcc tgtcagcgtg 1380
cctcgctacc agaggttccc actgggcgat gggcagtccc tcctcctcgc tgatgcccta 1440
cacacccaca gccccctctt cctggagggc agctcccggg acagcccgcc acttctggat 1500
gcccctgcct cgccccctca ggcctcgagg ttccagcgcc caggacggag gatgagcgag 1560
gggagctccc acagcgagag ctcggagtcc tcggacagca tggcacccgt gggtgcctcc 1620
cgcatcacag ccaagtggtg gggaagcaag aggatcgact atgccctgta ctgccctgat 1680
gtcctcacgg ccttccccac cgtggccctg ccccacctct tccacgccag ttactgggag 1740
tccacagacg tggtggcctt catcctgaga caggtaatgc gctatgagag cgtgaacatc 1800
aaggaaagcg cccgcctgga ccctgcagca ctgagtcctg ccaacccccg ggagaagtgg 1860
cttcgtaagc ggactcaggt caagctgagg aatgtcacgg ctaatcaccg ggccaatgat 1920
gtgattgctg ctgaagatgg cccccaggtc ctggtggggc ggttcatgta cgggcccctc 1980
gacatggtgg ctctgactgg agagaaggtg gacatcctag taatggcaga gccatcctca 2040
ggccgctggg tacacctgga cacagagatc accaacagca gtggtcgcat cacatacaat 2100
gtgccgcggc cccggcgcct gggggttggt gtctatcctg tgaagatggt cgtcaggggc 2160
gaccagacct gtgccatgag ctacctcacg gtgttgccca ggggcatgga gtgtgtagtg 2220
ttcagcattg atgggtcctt cgcggccagc gtgtctatca tgggaagcga ccccaaggtc 2280
cggccgggtg cagtggatgt tgtccggcac tggcaggact tgggctacat gatcctttac 2340
atcacgggac ggccggacat gcagaagcag cgggtggtgt cgtggctgtc ccagcacaac 2400
ttcccacagg gcatgatctt cttctccgat gggctggtgc atgacccgct gcggcagaag 2460
gccatcttcc tgcgcaacct catgcaggag tgcttcatca aaatcagtgc ggcctatggc 2520
tccacgaagg acatctctgt ctacagcgtg ctgggcctgc ctgcctccca gatcttcatt 2580
gtgggccggc ccaccaagaa gtaccaaacc cagtgccagt tcctgagcga gggctacgcc 2640
gcacacctgg ccgcgctgga ggccagccac cgctcacgcc caaagaagaa caactcgcgc 2700
atgatcctgc gcaagggcag cttcgggctg cacgcgcagc cagagttcct gcggaagcgc 2760
aaccacctgc gcagaaccat gtcagtgcag cagcccgacc cgcccgccgc caaccccaag 2820
cccgagcggg cccagagcca gcccgagtcg gacaaagacc acgagcggcc gctgccggcg 2880
ctcagctggg cgcgtgggcc ccccaagttc gagtcggtgc cctga 2925
<210> 3
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
aaagtcgctt acttactttg acaca 25
<210> 4
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ccctctggtc tctttagcca tcctg 25
<210> 5
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
cctcttacct cagttacaat ttata 25
<210> 6
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
atgggacaca tcgaccaaac a 21
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ttgcgacggc atcttggtag 20
Claims (4)
1. A method for constructing a BAVM model, comprising:
1) culturing the embryo of the zebra fish;
2) designing an inhibitor aiming at a homologous gene of PITPNM3 in zebra fish, wherein the inhibitor is morpholine antisense oligonucleotide and has a sequence shown in SEQ ID NO. 3;
3) 5ng of the inhibitor is injected into cells from a single cell stage to a two cell stage of a zebra fish embryo.
2. The application of a reagent for detecting mutant nucleotide in preparing a product for diagnosing BAVM is characterized in that compared with SEQ ID NO.1, the 274 th position of the mutant nucleotide detected by the reagent is mutated from C to T, and the mutated nucleotide sequence is shown as SEQ ID NO. 2.
3. The application of a reagent for detecting mutant protein in the preparation of products for diagnosing BAVM is characterized in that the mutant protein is coded by SEQ ID NO.2, and compared with the protein coded by SEQ ID NO.1, the 92 th amino acid of the mutant protein is mutated from Arg to a stop codon.
4. Use of a kit for detecting mutations in the manufacture of a product for diagnosing BAVM, said kit comprising a detection reagent comprising:
a) the detection reagent is characterized in that compared with the detected mutant nucleotide of SEQ ID NO.1, the 274 th position of the mutant nucleotide is mutated from C to T, and the mutated nucleotide sequence is shown as SEQ ID NO. 2;
b) and (3) a detection reagent, wherein the detected mutant protein is coded by SEQ ID NO.2, and compared with the protein coded by SEQ ID NO.1, the 92 th amino acid of the mutant protein is mutated from Arg to a stop codon.
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